Nitric oxide (NO) is an important cell signaling molecule that regulates blood pressure in endothelial cells, acts as an important molecule in macrophage cells for immune system defense against pathogens, and is a neurotransmitter in neuronal cells. Pathologically produced excesses of neuronal NO, however, have been correlated with almost all neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and Huntington's disease. The objective of this proposal is to diminish this excess neuronal NO by inhibition of the neuronal form of the enzyme that produces it, nitric oxide synthase (NOS). The successes we have had in the design of selective neuronal nitric oxide synthase (nNOS) inhibitors during the last funding period will be the springboard for future design and synthesis efforts, using crystallographic images of our inhibitors bound to NOS and structure-based computer design of new structures. At present some of our inhibitors have been found to be very effective at preventing cerebral palsy phenotype in a rabbit model for cerebral palsy. For this indication, administration by injection is acceptable. The important next step taken will be to enhance the oral bioavailability of these compounds while increasing their potency and selectivity for nNOS over the other two isoforms of NOS, endothelial NOS (eNOS) and inducible NOS (iNOS) to minimize side effects and toxicities. Up to this point we have been testing all of our compounds against lower animal nitric oxide synthases because they were the enzymes for which we could get crystal structure data from our collaborator, Professor Thomas Poulos. However, his group has recently been able to crystallize and resolve structures of human NOS isozymes. The focus in the new grant period will be on tuning selectivity for human nNOS over the other two human NOSs. In the last project period we have made two remarkable discoveries, both of which we will follow up in the upcoming project period. The first discovery was the finding by two of my collaborators, Professors Thomas Poulos and Victor Nizet, that some of our nNOS-selective inhibitors also inhibit bacterial NOS (bNOS) and are synergistic with antibiotics and other stress inducers in killing Gram-positive bacteria. In the new project period we will aim to identify bNOS inhibitors that are poor rat and/or human NOS inhibitors to attain selectivity for inhibition of bNOS. The second exciting discovery, by my collaborator Frank Meyskens, was that nitric oxide produced by nNOS in melanocytes is a significant cause for melanoma. Consequently, many of our nNOS-selective inhibitors were found to be effective anti-melanoma agents. However, there was little correlation between potency as rat nNOS inhibitors and their invasion potential in human metastatic melanoma cells. Although we plan to send our most potent and selective compounds to the Meyskens group for testing with metastatic melanoma, we also plan to identify proteins to which our compounds bind. We propose to identify cellular targets by photoaffinity labeling of proteins that bind to them; with he aid of click chemistry we will pull down bound proteins and identify what they are.